Search Results (224)

Chronic kidney disease (CKD) significantly affects canine health, but the precise cellular mechanisms of this condition remain elusive. In this study, we used single-cell RNA sequencing (scRNA-seq) to profile renal cellular gene expression in a canine model of X-linked hereditary nephropathy (XLHN). Dogs with this condition exhibit juvenile-onset CKD similar to that seen in human Alport syndrome. Post-mortem renal cortical tissues from an affected male dog and a heterozygous female dog were processed to obtain single-cell suspensions. In total, we recovered up to 13,190 cells and identified 11 cell types, including major kidney cells and immune cells. Differential gene expression analysis comparing the affected male and heterozygous female dogs identified cell-type specific pathways that differed in a subpopulation of proximal tubule cells. These pathways included the integrin signaling pathway and the pathway for inflammation mediated by chemokine and cytokine signaling. Additionally, using machine learning-empowered digital cytometry, we deconvolved bulk mRNA-seq data from a previous canine study, revealing changes in cell type proportions across CKD stages. These results underline the utility of single-cell methodologies and digital cytometry in veterinary nephrology. Full article

Background and Clinical Significance: Although dietary supplements have often been deemed safe, some have been linked to drug-induced nephropathy due to their diverse ingredients. The aim of this report is to enhance clinical awareness of a novel and emerging cause of Fanconi syndrome due to red yeast rice supplements and to contribute new histopathological and clinical data. Case Presentation: We report two cases of renal dysfunction and Fanconi syndrome associated with the use of red yeast rice supplements. Both patients presented with renal impairment accompanied by elevated markers of tubular injury, hypouricemia, hypokalemia, and glucosuria, consistent with Fanconi syndrome. Following the discontinuation of the red yeast rice supplement and initiation of steroid therapy, Fanconi syndrome resolved, however, moderate renal dysfunction persisted. Urinary NGAL levels improved after treatment in both cases. KIM-1 normalized in one case but remained elevated in the other. Uromodulin recovery was complete in one case and partial in the other. Renal biopsy revealed mild tubulointerstitial nephritis, with notable shedding of proximal tubular epithelial cells. Immunohistochemical analysis demonstrated reduced expression of URAT-1, Na-K ATPase, and Na-Pi IIa in some tubules. Conclusions: These findings suggest that renal injury induced by red yeast rice supplements is mediated by direct proximal tubular necrosis caused by a harmful substance in the supplement, resulting in persistence of tubular dysfunction. Full article

Background: Organic anion transporter 3 (OAT3) in the kidney proximal tubule cells plays a critical role in renal clearance of numerous endogenous metabolites and exogenous drugs and toxins. In this study, we discovered that epidermal growth factor (EGF) regulates the expression and activity of OAT3 through palmitoylation, a novel mechanism that has never been described in the OAT field. Methods/Results: Our results showed that treatment of OAT3-expressing cells with EGF led to a ~40% increase in OAT3 expression and OAT3-mediated transport of estrone sulfate, a prototypical substrate for OAT3. EGF-stimulated OAT3 transport activity was abrogated by H-89, a protein kinase A (PKA) inhibitor, indicating that an EGF-PKA signaling pathway is involved in the regulation of OAT3. We also showed that treatment of OAT3-expressing cells with EGF resulted in an enhancement of OAT3 palmitoylation, a novel type of post-translational modification for OATs, and such an enhancement was blocked by H-89, suggesting that the EGF-PKA signaling pathway participated in the modulation of OAT3 palmitoylation. Palmitoylation was catalyzed by a group of palmitoyltransfereases, and we showed that OAT3 palmitoylation and expression were inhibited by 2-BP, a general inhibitor for palmitoyltransfereases. We also explored the relationship among EGF/PKA signaling, OAT palmitoylation, and OAT transport activity. We treated OAT3-expressing cells with EGF or Bt2-cAMP, a PKA activator, in the presence and absence of 2-BP, followed by the measurement of OAT3-mediated transport of estrone sulfate. We showed that both EGF- and Bt2-cAMP-stimulated OAT3 transport activity were abolished by 2-BP, suggesting that palmitoylation mediates the regulation of EGF/PKA on OAT3. Finally, we showed that osimertinib, an anti-cancer drug/EGFR inhibitor, blocked EGF-stimulated OAT3 transport activity. Conclusions: In summary, we provided the first evidence that palmitoylation transduces the EGF/PKA signaling pathway to the modulation of OAT3 expression and function. Our study also provided an important implication that during comorbidity therapies, EGFR inhibitor drugs could potentially decrease the transport activity of renal OAT3, which would subsequently alter the therapeutic efficacy and toxicity of many co-medications that are OAT3 substrates. Full article

Arsenic, in the form of inorganic arsenite, is toxic to the kidney and can cause acute kidney injury, manifesting as destruction of proximal tubule cells. Nephron repair is possible through the proliferation of resident tubular progenitor cells expressing CD133 and CD24 surface markers. We simulated regenerative repair in the continued presence of i-As (III) using a cell culture model of a renal progenitor cell line expressing CD133 (PROM1) and CD24. Continued exposure and subculturing of progenitor cells to i-As (III) led to a reduction in the expression of PROM1 and CD24, as well as a decrease in the ability to differentiate into tubule-like structures. Cessation of i-As (III) and recovery for up to three passages resulted in continued repression of PROM1 and reduced ability to differentiate. Chronically exposed cells exhibited an ability to form colonies in soft agar, suggesting neoplastic transformation. Chronically exposed cells also exhibited an induction of CD44, a cell surface marker commonly found in renal cell carcinoma, as well as in tubular repair in chronic renal injury such as chronic kidney disease. These results demonstrate potential adverse outcomes of renal progenitor cells chronically exposed to a nephrotoxicant, as well as in environmental exposure to arsenic. Full article

Physiological control of blood pressure (BP) and extracellular fluid volume is mediated by the action of the renin-angiotensin system (RAS). The presence of RAS components throughout the nephron has been widely discussed. The (pro)renin receptor (PRR) is a member of the RAS widely expressed in the body of humans and rodents. In the kidney, PRR is expressed in mesangial cells, renal vasculature, and tubules of the proximal and distal nephron. Binding of the PRR to renin and prorenin promotes angiotensin (Ang) I-mediated sodium (Na⁺) reabsorption via the epithelial sodium channel (ENaC). The Goldblatt 2-kidney 1-clip (2K1C) is a model of experimental renovascular hypertension that displays activation of systemic and intrarenal RAS. We use the 2K1C hypertension mouse model for 7 days to evaluate the role of the PRR on renal αENaC expression, Na⁺ reabsorption, and BP using pharmacological systemic blockade of the PRR with PRO20 peptide. Mice undergoing or not to 2K1C surgery (0.13 mm clip internal gap) were chronically infused with PRO20 and compared to sham (control) mice to assess changes in systolic BP (SBP) and diastolic BP (DBP), intrarenal angiotensin-converting enzyme (ACE) activity, Ang II, and renal αENaC expression and natriuretic responses after a saline challenge. Renal artery obstruction increased SBP and DBP, intrarenal ACE activity, Ang II levels, Na⁺ retention, and αENaC expression in both kidneys. PRO20 prevented the increases in SBP, DBP, attenuated Na⁺ retention, and blunted intrarenal Ang II and αENaC levels in non-clipped kidneys of 2K1C mice. Chronic infusion of the PRR for 7 days prevents hypertensive responses in part due to impaired αENaC upregulation and intrarenal Ang II formation in the early phase of the development of renovascular hypertension in 2K1C Goldblatt mice. Full article

In this work, the utilization of calcium and strontium by the (Ca²⁺ + Mg²⁺)ATPase of the basolateral plasma membrane of renal proximal convoluted tubules were compared. [⁹⁰Sr]Sr²⁺ and [⁴⁵Ca]Ca²⁺ uptake by vesicles derived from this membrane were strictly dependent on ATP and Mg²⁺, and no other nucleotide was able to support the transport. Each cation inhibited the uptake of the other one in a purely competitive fashion (the same Vmax; increased K_0.5), without causing a significant change in the influx rate. These results indicate that both cations bind at the same transport site on the enzyme, facing the cytosolic surface of the cell. The K_0.5 for Sr²⁺ obtained for (Sr²⁺ + Mg²⁺)ATPase activity was 13.1 ± 0.2 µM and for Sr²⁺ uptake was 13.4 ± 0.1 µM. They were higher than K_0.5 for Ca²⁺ obtained for (Ca²⁺ + Mg²⁺)ATPase activity (0.42 ± 0.03 µM) and for Ca²⁺ uptake (0.28 ± 0.02 µM). It is postulated that the lower ATPase affinity for Sr²⁺ is associated with greater steric difficulties for the occupation by this cation of the binding and transport sites, as a consequence of its greater crystal ionic radius (1.13 Å for Sr²⁺ against 0.99 Å for Ca²⁺). Full article

Primary mouse renal proximal tubule epithelial cells (moRPTECs) were immortalized by lentivirus transduction to create hTERT or SV40LgT (LgT) cell lines. Prior work showed a more pronounced injury and repair response in LgT versus hTERT cells after chemical challenge. We hypothesized that unique genomic changes occurred after immortalization, altering critical genes and pathways. RNA-seq profiling and whole-genome sequencing (WGS) of parent, hTERT, and LgT cells showed that 92.5% of the annotated transcripts were shared, suggesting a conserved proximal tubule expression pattern. However, the cell lines exhibited unique transcriptomic and genomic profiles different from the parent cells. Three transcript classes were quite relevant for chemical challenge response—Cyps, ion channels, and metabolic transporters—each important for renal function. A pathway analysis of the hTERT cells suggested alterations in intermediary and energy metabolism. LgT cells exhibited pathway activation in cell cycle and DNA repair that was consistent with replication stress. Genomic karyotyping by combining WGS and RNA-seq data showed increased gene copy numbers in chromosome 5 for LgT cells, while hTERT cells displayed gene copy losses in chromosomes 4 and 9. These data suggest that the exaggerated transcriptional responses of LgT cells versus hTERT cells result from differences in gene copy numbers, replication stress, and the unique selection processes underlying LgT or hTERT immortalization. Full article

Lipotoxicity, resulting from the buildup of excess lipids in non-adipose tissues, is increasingly recognized as a major contributor to the progression of kidney disease, highlighting the need for alternative models to assess its effects on renal cells. The main aim of this study was to investigate the usefulness of Caki-1, a human proximal tubule (PT) and renal cell carcinoma (RCC) representative cell line, as a 3D model system for studying free fatty acid-induced PT lipotoxicity. Caki-1 spheroids were generated and maintained on ultra-low attachment plates and characterized regarding time-dependent morphology changes. In optimal 3D culture conditions, Caki-1 cells formed well-defined large compact spheroids with uniform morphology, good circularity, and increased diameter from days 4–12. Chronic exposure to saturated palmitate resulted in dose- and time-dependent spheroid disintegration and cell death, including dispersed and flattened spheroid morphology, with increased dead cells in the peripheral layers and decreased spheroid core. Moreover, palmitate-treated spheroids showed a significant increase in cleaved poly(ADP-ribose) polymerase (PARP) and active caspase-3. Palmitate-induced PARP cleavage, as well as endoplasmic reticulum (ER) stress and autophagy dysfunction, were blunted by triacsin C, an inhibitor of long-chain acyl-CoA synthetases. In addition, co-incubation with unsaturated oleate prevented palmitate-induced spheroid disintegration and apoptotic cell death in Caki-1 3D culture. While fatty acid overload upregulated lipid droplet protein perilipin 2 in Caki-1 cells, knockdown of perilipin 2 by siRNAs resulted in an exacerbation of palmitate-induced cell death. Together, these results indicate that the 3D Caki-1 spheroid model is a simple and reproducible in vitro system for studying renal lipotoxicity and lipid metabolism that gives useful readouts at the molecular, cellular, and multicellular levels. Full article

Background/Objectives: The kidneys are essential for eliminating drugs and chemicals from the human body and renal epithelial cells are particularly vulnerable to damage caused by xenobiotics and their metabolites. Drug-induced kidney toxicity is a major cause of drug attrition during preclinical and clinical development and the ability to predict renal toxicity remains a pressing challenge, necessitating more predictive in vitro models. However, the abundance of commercially available renal proximal tubule epithelial cell (RPTEC) sources complicates the selection of the most predictive cell types. Methods: This study compared a wide range of RPTEC sources, including primary cells (Lonza) and various RPTEC lines from different vendors, such as ciPTECs (Cell4Pharma), TERT1/RPTECs (ATCC), and HEK293 (GenoMembrane), including OAT1-overexpressing variants. HepG2 cells were included for a comparison of organ specificity. The different cells were cultured in 96- or 384-well plates and exposed to 12 drugs for 72 h at a concentration yielding a response (0.3–300 µM) to evaluate their ability to predict clinical outcomes. The CellTiterGlo^® assay was used to measure cell viability, and transcriptome data from unexposed cells was analyzed using the TempO-seq^® S1500+ platform. Results: Gene expression data showed that the primary kidney cells most closely matched the transcriptome of the human kidney medulla, followed by the TERT1 and ciPTEC lines, with the HEK lines showing the lowest similarity. The RPTEC sources showed clustering by cell type, with OAT1 overexpression driving changes in metabolic, detoxification, and immune pathways, especially in TERT1 cells. Cell viability data were used to determine points of departure (PODs) which were compared to human serum Cmax values to assess safety margins. The TERT1 and ciPTEC RPTEC lines demonstrated the highest predictive performance for nephrotoxicity, with OAT1 overexpression significantly enhancing sensitivity, accuracy, and overall predictive power (MCC scores: 0.764 and 0.667, respectively). In contrast, HepG2 cells showed the lowest performance across all metrics, highlighting the critical role of cell type and transporter expression in nephrotoxicity prediction. Conclusions: This study highlights important differences among RPTEC sources and their utility in drug safety studies of the renal proximal tubule. We show that while improved cell options for renal proximal tubule are needed, OAT1-overexpressing RPTECs are a superior model to the background cell type. Full article

Background: Sorting nexin 19 (SNX19) is important in the localization and trafficking of the dopamine D₁ receptor (D₁R) to lipid raft microdomains. However, the interaction between SNX19 and the lipid raft components caveolin-1 or flotillin-1 and, in particular, their roles in the cellular endocytosis and cell membrane trafficking of the D₁R have not been determined. Methods: Caveolin-1 and flotillin-1 motifs were analyzed by in silico analysis; colocalization was observed by confocal immunofluorescence microscopy; protein-protein interaction was determined by co-immunoprecipitation. Results: In silico analysis revealed the presence of putative caveolin-1 and flotillin-1 binding motifs within SNX19. In mouse and human renal proximal tubule cells (RPTCs), SNX19 was localized mainly in lipid rafts. In mouse RPTCs transfected with wild-type (WT) Snx19, fenoldopam (FEN), a D₁-like receptor agonist, increased the colocalization of SNX19 with caveolin-1 and flotillin-1. FEN also increased the co-immunoprecipitation of SNX19 with caveolin-1 and flotillin-1, effects that were prevented by SCH39166, a D₁-like receptor antagonist. The FEN-mediated increase in the residence of SNX19 in lipid rafts and the colocalization of the D₁R with caveolin-1 and flotilin-1 were attenuated by the deletion of a caveolin-1 (YHTVNRRYREF) (ΔCav1) or a flotillin-1 (EEGPGTETETGLPVS) (ΔFlot1) binding motif. The FEN-mediated increase in intracellular cAMP production was also impaired by the deletion of either the flotillin-1 or caveolin-1 binding motif. Nocodazole, a microtubule depolymerization inhibitor, interfered with the FEN-mediated increase in the colocalization between SNX19 and D₁R. Conclusion: SNX19 contains caveolin-1 and flotillin-1 binding motifs, which play an important role in D₁R endocytosis and signaling. Full article

Cisplatin (cis-diclorodiamminoplatin, CDDP) is a common chemotherapeutic agent for solid tumors, but its use is limited by severe side effects such as ototoxicity and nephrotoxicity. Variability in CDDP dosing and administration, along with high mortality and sensitivity in animal models, complicates experimental approaches. This study aimed to evaluate ototoxic damage in rats by comparing a single bolus versus three divided CDDP injections, also considering nephrotoxic effects. Twenty-four Sprague-Dawley rats were divided into three groups: eight received a single intraperitoneal injection of CDDP (14 mg/kg), eight received three injections (4.6 mg/kg/day), and eight were untreated controls. All CDDP-treated rats showed significant high-frequency hearing loss and morphological damage, including cochlear outer hair cell loss and renal glomerular atrophy with proximal tubule necrosis. Oxidative stress markers (nitrotyrosine and SOD1 expression) confirmed cochlear and renal alterations. Notably, the single bolus group had a 25% mortality rate and significant weight loss among survivors, unlike the other groups. This study introduces the novel finding that cumulative administration of three doses reduces mortality and weight loss while maintaining similar ototoxic and nephrotoxic effects. Therefore, cumulative administration is recommended for future studies to mitigate ototoxic and nephrotoxic damage, offering a potentially improved protocol for the administration of cisplatin. Full article

Cisplatin is a commonly used chemotherapeutic agent in the treatment of a wide array of cancers. Due to its active transport into the kidney proximal tubule cells, cisplatin treatment can cause a buildup of this nephrotoxic compound in the kidney, resulting in acute kidney injury (AKI). About 30% of patients receiving cisplatin chemotherapy develop cisplatin-induced AKI. JP4-039 is a mitochondria-targeted reactive oxygen species (ROS) and electron scavenger. Recent studies have shown that JP4-039 mitigates a variety of genotoxic insults in preclinical studies in rodents by suppressing oxidative stress-mediated tissue damage and blocking apoptosis and ferroptosis. However, the benefits of JP4-039 treatment have not been tested in the setting of AKI. In this study, we investigated the potential renoprotective effect of JP4-039 on cisplatin-induced AKI. To address this goal, we treated mice with JP4-039 before or after cisplatin administration and analyzed them for functional and molecular changes in the kidney. JP4-039 co-administration attenuated cisplatin-induced renal dysfunction and histopathological changes. Upregulation of tubular injury markers was also suppressed by JP4-039. Mechanistically, JP4-039 suppressed lipid peroxidation, prevented tissue oxidative stress, and preserved the glutathione levels in cisplatin-injected mice. An increase in cisplatin-induced apoptosis and ferroptosis was also alleviated by the compound. Moreover, JP4-039 inhibited cytokine overproduction in cisplatin-injected mice. Together, our findings demonstrate that JP4-039 is a promising therapeutic agent against cisplatin-induced kidney injury. Full article

Renal cell carcinoma (RCC) is the most common form of kidney cancer, known for its immune evasion and resistance to chemotherapy. Evidence indicates that the SARS-CoV-2 virus may worsen outcomes for RCC patients, as well as patients with diminished renal function. Evidence suggests that the SARS-CoV-2 virus may exacerbate outcomes in RCC patients and those with impaired renal function. This study explored the unidirectional effects of RCC cells and the SARS-CoV-2 spike protein (S protein) on human renal proximal tubule epithelial cells (RPTECs) using a microphysiological approach. We co-cultured RCC cells (Caki-1) with RPTEC and exposed them to the SARS-CoV-2 S protein under dynamic 3D conditions. The impact on metabolic activity, gene expression, immune secretions, and S protein internalization was evaluated. The SARS-CoV-2 S protein was internalized by RPTEC but poorly interacted with RCC cells. RPTECs exposed to RCC cells and the S protein exhibited upregulated expression of genes involved in immunogenic pathways, particularly those related to antigen processing and presentation via the major histocompatibility complex I (MHCI). Additionally, increased TNF-α secretion suggested a pro-inflammatory response. Metabolic shifts toward glycolysis were observed in RCC co-culture, while the presence of the S protein led to minor changes. The presence of RCC cells amplified the immune-modulatory effects of the SARS-CoV-2 S protein on the renal epithelium, potentially exacerbating renal inflammation and fostering tumor-supportive conditions. These findings suggest that COVID-19 infections can impact renal function in the presence of kidney cancer. Full article

Cisplatin (CisPt) is a widely used chemotherapeutic agent. However, its nephrotoxic effects pose significant risks, particularly for the development of acute kidney injury (AKI) and potential progression to chronic kidney disease (CKD). The present study investigates the impact of non-lethal exposure of CisPt to immortalized human renal epithelial precursor TERT cells (HRTPT cells) that co-express PROM1 and CD24, markers characteristic of renal progenitor cells. Over eight serial passages, HRTPT cells were exposed to 1.5 µM CisPt, leading to an initial growth arrest, followed by a gradual recovery of proliferative capacity. Despite maintaining intracellular platinum (Pt) levels, the cells exhibited normal morphology by passage eight (P8), with elevated expression of renal stress and damage markers. However, the ability to form domes was not restored. RNA-seq analysis revealed 516 differentially expressed genes between CisPt-exposed and control cells, with significant correlations to cell cycle and adaptive processes, as determined by the Reactome, DAVID, and Panther analysis programs. The progenitor cells treated with CisPt displayed no identity, or close identity, with cells of the normal human nephron. Additionally, several upregulated genes in P8 cells were linked to cancer cell lines, suggesting a complex interaction between CisPt exposure and cellular repair mechanisms. In conclusion, our study demonstrates that renal progenitor cells can recover from CisPt exposure and regain proliferative potential in the continued presence of both extracellular CisPt and intracellular Pt. Full article

Background: Fanconi syndrome is a disorder of renal proximal tubule transport characterized by metabolic acidosis, amino aciduria, glucosuria, and phosphaturia. There are acquired and hereditary forms of this disorder. A late-onset form of Fanconi syndrome in Basenjis was first described in 1976 and is now recognized as an inherited disease in these dogs. In part because of the late onset of disease signs, the disorder has not been eradicated from the breed by selective mating. A study was therefore undertaken to identify the molecular genetic basis of the disease so that dogs could be screened prior to breeding in order to avoid generating affected offspring. Methods: Linkage analysis within a large family of Basenjis that included both affected and unaffected individuals was performed to localize the causative variant within the genome. Significant linkage was identified between chromosome 3 (CFA3) makers and the disease phenotype. Fine mapping restricted the region to a 2.7 Mb section of CFA3. A whole genome sequence of a Basenji affected with Fanconi syndrome was generated, and the sequence data were examined for the presence of potentially deleterious homozygous variants within the mapped region. Results: A homozygous 317 bp deletion was identified in the last exon of FAN1 of the proband. 78 Basenjis of known disease status were genotyped for the deletion variant. Among these dogs, there was almost complete concordance between genotype and phenotype. The only exception was one dog that was homozygous for the deletion variant but did not exhibit signs of Fanconi syndrome. Conclusions: These data indicate that the disorder is very likely the result of FAN1 deficiency. The mechanism by which this deficiency causes the disease signs remains to be elucidated. FAN1 has endonuclease and exonuclease activity that catalyzes incisions in regions of double-stranded DNA containing interstrand crosslinks. FAN1 inactivation may cause Fanconi syndrome in Basenjis by sensitization of kidney proximal tubule cells to toxin-mediated DNA crosslinking, resulting in the accumulation of genomic and mitochondrial DNA damage in the kidney. Differential exposure to environmental toxins that promote DNA crosslink formation may explain the wide age-at-onset variability for the disorder in Basenjis. Full article